Packaging systems for human recombinant adenovirus to be used in gene therapy

ABSTRACT

Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces adenovirus incapable of replicating. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of Ser. No. 09/506,548 filedFeb. 16, 2000 now U.S. Pat. No. 6,602,706, which is a divisional of Ser.No. 09/334,765 filed Jun. 16, 1999 now U.S. Pat. No. 6,238,893, which isa continuation of Ser. No. 08/793,170 filed Mar. 25, 1997, now U.S. Pat.No.5,994,128, which is the ational stage and claims priority toInternational Patent Application PCT/NL96/00244 filed on Jun. 14, 1996,which itself claims priority from European patent application 95201728.3filed on Jun. 26, 1995, and European patent application 95201611.1 filedon Jun. 15, 1995.

TECHNICAL FIELD

The invention relates to the field of recombinant DNA technology, morein particular to the field of gene therapy. In particular the inventionrelates to gene therapy using materials derived from adenovirus,specifically human recombinant adenovirus. It especially relates tonovel virus derived vectors and novel packaging cell lines for vectorsbased on adenoviruses.

BACKGROUND

Gene therapy is a recently developed concept for which a wide range ofapplications can be and have been envisioned. In gene therapy a moleculecarrying genetic information is introduced into some or all cells of ahost, as a result of which the genetic information is added to the hostin a functional format.

The genetic information added may be a gene or a derivative of a gene,such as a cDNA, which encodes a protein. This is a functional format inthat the protein can be expressed by the machinery of the host cell.

The genetic information can also be a sequence of nucleotidescomplementary to a sequence of nucleotides (either DNA or RNA) presentin the host cell. This is a functional format in that the added DNA(nucleic acid) molecule or copies made thereof in situ are capable ofbase pairing with the complementary sequence present in the host cell.

Applications include the treatment of genetic disorders by supplementinga protein or other substance which, because of the genetic disorder, iseither absent or present in insufficient amounts in the host, thetreatment of tumors and the treatment of other acquired diseases such as(auto)immune diseases, infections, etc.

As may be inferred from the foregoing, three basic different approachesexist in gene therapy: the first directed towards compensating for adeficiency in a (mammalian) host, the second directed towards theremoval or elimination of unwanted substances (organisms or cells) andthe third towards application of a recombinant vaccine (against tumorsor foreign micro-organisms).

For the purpose of gene therapy, adenoviruses carrying deletions havebeen proposed as suitable vehicles for genetic information. Adenovirusesare non-enveloped DNA viruses. Gene-transfer vectors derived fromadenoviruses (so-called “adenoviral vectors”) have a number of featuresthat make them particularly useful for gene transfer for such purposes.For example, the biology of the adenoviruses is characterized in detail,the adenovirus is not associated with severe human pathology, the virusis extremely efficient in introducing its DNA into the host cell, thevirus can infect a wide variety of cells and has a broad host-range, thevirus can be produced in large quantities with relative ease, and thevirus can be rendered replication defective by deletions in theearly-region 1 (“E1”) of the viral genome.

The adenovirus genome is a linear double-stranded DNA molecule ofapproximately 36000 base pairs with the 55-kDa terminal proteincovalently bound to the 5′ terminus of each strand. The adenoviral(“Ad”) DNA contains identical Inverted Terminal Repeats (“ITR”) of about100 base pairs with the exact length depending on the serotype. Theviral origins of replication are located within the ITRs exactly at thegenome ends. DNA synthesis occurs in two stages. First, the replicationproceeds by strand displacement, generating a daughter duplex moleculeand a parental displaced strand. The displaced strand is single strandedand can form a so-called “panhandle” intermediate, which allowsreplication initiation and generation of a daughter duplex molecule.Alternatively, replication may proceed from both ends of the genomesimultaneously, obviating the requirement to form the panhandlestructure. The replication is summarized in FIG. 14 adapted from Lechnerand Kelly (1977).

During the productive infection cycle, the viral genes are expressed intwo phases: the early phase, which is the period up to viral DNAreplication, and the late phase, which coincides with the initiation ofviral DNA replication. During the early phase only the early geneproducts, encoded by regions E1, E2, E3 and E4, are expressed, whichcarry out a number of functions that prepare the cell for synthesis ofviral structural proteins (Berk, 1986). During the late phase the lateviral gene products are expressed in addition to the early gene productsand host cell DNA and protein synthesis are shut off. Consequently, thecell becomes dedicated to the production of viral DNA and of viralstructural proteins (Tooze, 1981).

The E1 region of adenovirus is the first region of adenovirus expressedafter infection of the target cell. This region consists of twotranscriptional units, the E1A and E1B genes, which both are requiredfor oncogenic transformation of primary (embryonic) rodent cultures. Themain functions of the E1A gene products are 1) to induce quiescent cellsto enter the cell cycle and resume cellular DNA synthesis, and 2) totranscriptionally activate the E1B gene and the other early regions (E2,E3, E4). Transfection of primary cells with the E1A gene alone caninduce unlimited proliferation (immortalization), but does not result incomplete transformation. However, expression of E1A in most casesresults in induction of programmed cell death (apoptosis), and onlyoccasionally immortalization (Jochemsen et al., 1987). Co-expression ofthe E1B gene is required to prevent induction of apoptosis and forcomplete morphological transformation to occur. In established immortalcell lines, high level expression of E1A can cause completetransformation in the absence of E1B (Roberts et al., 1985).

The E1B encoded proteins assist E1A in redirecting the cellularfunctions to allow viral replication. The E1B 55 kD and E4 33kDproteins, which form a complex that is essentially localized in thenucleus, function in inhibiting the synthesis of host proteins and infacilitating the expression of viral genes. Their main influence is toestablish selective transport of viral mRNAs from the nucleus to thecytoplasm, concomittantly with the onset of the late phase of infection.The E1B 21 kD protein is important for correct temporal control of theproductive infection cycle, thereby preventing premature death of thehost cell before the virus life cycle has been completed. Mutant virusesincapable of expressing the E1B 21 kD gene-product exhibit a shortenedinfection cycle that is accompanied by excessive degradation of hostcell chromosomal DNA (deg-phenotype) and in an enhanced cytopathiceffect (cyt-phenotype) (Telling et al, 1994). The deg and cyt phenotypesare suppressed when in addition the E1A gene is mutated, indicating thatthese phenotypes are a function of E1A (White et al., 1988).Furthermore, the E1B 21 kDa protein slows down the rate by which E1Aswitches on the other viral genes. It is not yet known through whichmechanisms E1B 21 kD quenches these E1A dependent functions.

Vectors derived from human adenoviruses, in which at least the E1 regionhas been deleted and replaced by a gene of interest, have been usedextensively for gene therapy experiments in the pre-clinical andclinical phase.

As stated before, all adenovirus vectors currently used in gene therapyare believed to have a deletion in the E1 region, where novel geneticinformation can be introduced. The E1 deletion renders the recombinantvirus replication defective (Stratford-Perricaudet and Perricaudet,1991). We have demonstrated that recombinant adenoviruses are able toefficiently transfer recombinant genes to the rat liver and airwayepithelium of rhesus monkeys (Bout et al., 1994b; Bout et al., 1994a).In addition, we (Vincent et al., 1996a; Vincent et al., 1996b) andothers (see, e.g., Haddada et al., 1993) have observed a very efficientin vivo adenovirus mediated gene transfer to a variety of tumor cells invitro and to solid tumors in animals models (lung tumors, glioma) andhuman xenografts in immunodeficient mice (lung) in vivo (reviewed byBlaese et al., 1995).

In contrast to (for instance) retroviruses, adenoviruses 1) do notintegrate into the host cell genome, 2) are able to infect non-dividingcells, and 3) are able to efficiently transfer recombinant genes in vivo(Brody and Crystal, 1994). Those features make adenoviruses attractivecandidates for in vivo gene transfer of, for instance, suicide orcytokine genes into tumor cells.

However, a problem associated with current recombinant adenovirustechnology is the possibility of unwanted generation of replicationcompetent adenovirus (“RCA”) during the production of recombinantadenovirus (Lochmüller et al., 1994; Imler et al., 1996). This is causedby homologous recombination between overlapping sequences from therecombinant vector and the adenovirus constructs present in thecomplementing cell line, such as the 293 cells (Graham et al., 1977).RCA in batches to be used in clinical trials is unwanted because RCA 1)will replicate in an uncontrolled fashion, 2) can complement replicationdefective recombinant adenovirus, causing uncontrolled multiplication ofthe recombinant adenovirus and 3) batches containing RCA inducesignificant tissue damage and hence strong pathological side effects(Lochmüller et al., 1994). Therefore, batches to be used in clinicaltrials should be proven free of RCA (Ostrove, 1994).

It was generally thought that E1-deleted vectors would not express anyother adenovirus genes. However, recently it has been demonstrated thatsome cell types are able to express adenovirus genes in the absence ofE1 sequences. This indicates, that some cell types possess the machineryto drive transcription of adenovirus genes. In particular, it wasdemonstrated that such cells synthesize E2A and late adenovirusproteins.

In a gene therapy setting, this means that transfer of the therapeuticrecombinant gene to somatic cells not only results in expression of thetherapeutic protein but may also result in the synthesis of viralproteins. Cells that express adenoviral proteins are recognized andkilled by Cytotoxic T Lymphocytes, which thus 1) eradicates thetransduced cells and 2) causes inflammations (Bout et al., 1994a;Engelhardt et al., 1993; Simon et al., 1993). As this adverse reactionis hampering gene therapy, several solutions to this problem have beensuggested, such as 1) using immunosuppressive agents after treatment; 2)retainment of the adenovirus E3 region in the recombinant vector (seepatent application EP 95202213) and 3) and using temperature sensitive(“ts”) mutants of human adenovirus, which have a point mutation in theE2A region rendering them temperature sensitive, as has been claimed inpatent WO/28938.

However, these strategies to circumvent the immune response have theirlimitations. The use of ts mutant recombinant adenovirus diminishes theimmune response to some extent, but was less effective in preventingpathological responses in the lungs (Engelhardt et al., 1994a).

The E2A protein may induce an immune response by itself and it plays apivotal role in the switch to the synthesis of late adenovirus proteins.Therefore, it is attractive to make temperature sensitive recombinanthuman adenoviruses.

A major drawback of this system is the fact that, although the E2protein is unstable at the non-permissive temperature, the immunogenicprotein is still being synthesized. In addition, it is to be expectedthat the unstable protein does activate late gene expression, albeit toa low extent. ts125 mutant recombinant adenoviruses have been tested,and prolonged recombinant gene expression was reported (Yang et al.,1994b; Engelhardt et al., 1994a; Engelhardt et al., 1994b; Yang et al,1995). However, pathology in the lungs of cotton rats was still high(Engelhardt et al., 1994a), indicating that the use of ts mutantsresults in only a partial improvement in recombinant adenovirustechnology. Others (Fang et al, 1996) did not observe prolonged geneexpression in mice and dogs using ts125 recombinant adenovirus. Anadditional difficulty associated with the use of ts 125 mutantadenoviruses is that a high frequency of reversion is observed. Theserevertants are either real revertants or the result of second sitemutations (Kruijer et al., 1983; Nicolas et al, 1981). Both types ofrevertants have an E2A protein that functions at normal temperature andhave therefore similar toxicity as the wild-type virus.

SUMMARY OF THE INVENTION

In one aspect of the invention, this problem in virus production issolved in that we have developed packaging cells that have nooverlapping sequences with a new basic vector and thus are suited forsafe large scale production of recombinant adenoviruses one of theadditional problems associated with the use of recombinant adenovirusvectors is the host-defense reaction against treatment with adenovirus.

Briefly, recombinant adenoviruses are deleted for the E1 region. Theadenovirus E1 products trigger the transcription of the other earlygenes (E2, E3, E4), which consequently activate expression of the latevirus genes.

In another aspect of the present invention we therefore delete E2Acoding sequences from the recombinant adenovirus genome and transfectthese E2A sequences into the (packaging) cell lines containing E1sequences to complement recombinant adenovirus vectors.

Major hurdles in this approach are 1) that E2A should be expressed tovery high levels and 2) that E2A protein is very toxic to cells.

The current invention in yet another aspect therefore discloses use ofthe ts125 mutant E2A gene, which produces a protein that is not able tobind DNA sequences at the non permissive temperature. High levels ofthis protein may be maintained in the cells (because it is non-toxic atthis temperature) until the switch to the permissive temperature ismade. This can be combined with placing the mutant E2A gene under thedirection of an inducible promoter, such as for instance tet,methallothionein, steroid inducible promoter, retinoic acid β-receptoror other inducible systems. However in yet another aspect of theinvention, the use of an inducible promoter to control he moment ofproduction of toxic wild-type E2A is disclosed.

Two salient additional advantages of E2A-deleted recombinant adenovirusare the increased capacity to harbor heterologous sequences and thepermanent selection for cells that express the mutant E2A. This secondadvantage relates to the high frequency of reversion of ts125 mutation:when reversion occurs in a cell line harboring ts125 E2A, this will belethal to the cell. Therefore, there is a permanent selection for thosecells that express the ts125 mutant E2A protein. In addition, as we inone aspect of the invention generate E2A-deleted recombinant adenovirus,we will not have the problem of reversion in our adenoviruses.

In yet another aspect of the invention as a further improvement the useof non-human cell lines as packaging cell lines is disclosed.

For GMP production of clinical batches of recombinant viruses it isdesirable to use a cell line that has been used widely for production ofother biotechnology products. Most of the latter cell lines are frommonkey origin, which have been used to produce, for example, vaccines.

These cells can not be used directly for the production of recombinanthuman adenovirus, as human adenovirus cannot replicate, or replicatesonly to low levels in cells of monkey origin. A block in the switch ofearly to late phase of adenovirus lytic cycle underlies defectivereplication. However, host range mutations in the human adenovirusgenome are described (hr400-404) which allow replication of humanviruses in monkey cells. These mutations reside in the gene encoding E2Aprotein (Klessig and Grodzicker, 1979; Klessig et al., 1984; Rice andKlessig, 1985)(Klessig et al., 1984). Moreover, mutant viruses have beendescribed that harbor both the hr and temperature-sensitive ts125phenotype (Brough et al., 1985; Rice and Klessig, 1985).

We therefore generate packaging cell lines of monkey origin (e.g., VERO,CV1) that harbor:

1) E1 sequences, to allow replication of E1/E2 defective adenoviruses,and

2) E2A sequences, containing the hr mutation and the ts125 mutation,named ts400 (Brough et al., 1985; Rice and Klessig, 1985) to preventcell death by E2A overexpression, and/or

3) E2A sequences, just containing the hr mutation, under the control ofan inducible promoter, and/or

4) E2A sequences, containing the hr mutation and the ts125 mutation(ts400), under the control of an inducible promoter

Furthermore we disclose the construction of novel and improvedcombinations of packaging cell lines and recombinant adenovirus vectors.We provide:

1) a novel packaging cell line derived from diploid human embryonicretinoblasts (“HER”) that harbors nt. 80-5788 of the AdS genome. Thiscell line, named 911, deposited under no 95062101 at the ECACC, has manycharacteristics that make it superior to the commonly used 293 cells(Fallaux et al., 1996).

2) novel packaging cell lines that express just E1A genes and not E1Bgenes. Established cell lines (and not human diploid cells of which 293and 911 cells are derived) are able to express E1A to high levelswithout undergoing apoptotic cell death, as occurs in human diploidcells that express E1A in the absence of E1B. Such cell lines are ableto trans-complement E1B-defective recombinant adenoviruses, becauseviruses mutated for E1B21 kD protein are able to complete viralreplication even faster than wild-type adenoviruses (Telling et al.,1994). The constructs are described in detail below, and graphicallyrepresented in FIGS. 1-5. The constructs are transfected into thedifferent established cell lines and are selected for high expression ofE1A. This is done by operatively linking a selectable marker gene (e.g.NEO gene) directly to the E1B promoter. The E1B promoter istranscriptionally activated by the E1A gene product and thereforeresistance to the selective agent (e.g., G418 in the case NEO is used asthe selection marker) results in direct selection for desired expressionof the E1A gene.

3) Packaging constructs that are mutated or deleted for E1B21 kD, butjust express the 55 kD protein.

4) Packaging constructs to be used for generation of complementingpackaging cell lines from diploid cells (not exclusively of humanorigin) without the need for selection with marker genes. These cellsare immortalized by expression of E1A. However, in this particular caseexpression of E1B is essential to prevent apoptosis induced by E1Aproteins. Selection of E1 expressing cells is achieved by selection forfocus formation (immortalization), as described for 293 cells (Graham etal., 1977) and 911 cells (Fallaux et al., 1996), that are E1-transformedhuman embryonic kidney (“HEK”) cells and human embryonic retinoblasts(“HER”), respectively.

5) After transfection of HER cells with construct pIG.E1B (FIG. 4),seven independent cell lines could be established. These cell lines weredesignated PER.C1, PER.C3, PER.C4, PER.C5, PER.C6, PER.C8 and PER.C9.PER denotes PGK-E1-Retinoblasts. These cell lines express E1A and E1Bproteins, are stable (e.g, PER.C6 for more than 57 passages) andcomplement E1 defective adenovirus vectors. Yields of recombinantadenovirus obtained on PER cells are a little higher than obtained on293 cells. One of these cell lines (PER.C6) has been deposited at theECACC under number 96022940.

6) New adenovirus vectors with extended E1 deletions (deletion nt.459-3510). Those viral vectors lack sequences homologous to E1 sequencesin said packaging cell lines. These adenoviral vectors contain pIXpromoter sequences and the pIX gene, as pIX (from its natural promotersequences) can only be expressed from the vector and not by packagingcells (Matsui et al., 1986, Hoeben and Fallaux, pers.comm.; Imler etal., 1996).

7) E2A expressing packaging cell lines preferably based on either E1Aexpressing established cell lines or E1A-E1B expressing diploid cells(see under 2-4). E2A expression is either under the control of aninducible promoter or the E2A ts125 mutant is driven by either aninducible or a constitutive promoter.

8) Recombinant adenovirus vectors as described before (see 6) butcarrying an additional deletion of E2A sequences.

9) Adenovirus packaging cells from monkey origin that are able totrans-complement E1-defective recombinant adenoviruses. They arepreferably co-transfected with pIG.E1AE1B and pIG.NEO, and selected forNEO resistance. Such cells expressing E1 A and E1 B are able totranscomplement E1 defective recombinant human adenoviruses, but will doso inefficiently because of a block of the synthesis of late adenovirusproteins in cells of monkey origin (Klessig and Grodzicker, 1979). Toovercome this problem, we generate recombinant adenoviruses that harbora host-range mutation in the E2A gene, allowing human adenoviruses toreplicate in monkey cells. Such viruses are generated as described inFIG. 12, except DNA from a hr-mutant is used for homologousrecombination.

10) Adenovirus packaging cells from monkey origin as described under 9,except that they will also be co-transfected with E2A sequencesharboring the hr mutation. This allows replication of human adenoviruseslacking E1 and E2A (see under 8). E2A in these cell lines is eitherunder the control of an inducible promoter or the tsE2A mutant is used.In the latter case, the E2A gene will thus carry both the ts mutationand the hr mutation (derived from ts400). Replication competent humanadenoviruses have been described that harbor both mutations (Brough etal., 1985; Rice and Klessig, 1985).

A further aspect of the invention provides otherwise improved adenovirusvectors, as well as novel strategies for generation and application ofsuch vectors and a method for the intracellular amplification of linearDNA fragments in mammalian cells.

BRIEF DESCRIPTION OF THE FIGURES

The following figures and drawings may help to understand the invention:

FIG. 1 illustrates the construction of pBS.PGK.PCRI.

FIG. 2 illustrates the construction of pIG.E1A.E1B.X.

FIGS. 3A and 3B illustrate the construction of pIG.E1A.NEO.

FIG. 4 illustrates the construction of pIG.E1A.E1B.

FIG. 5 illustrates the construction of pIG.NEO.

FIG. 6 illustrates the transformation of primary baby rat kidney (“BRK”)cells by adenovirus packaging constructs.

FIG. 7 illustrates a Western blot analysis of A549 clones transfectedwith pIG.E1A.NEO and HER cells transfected with pIG.E1A.E1B (PERclones).

FIG. 8 illustrates a Southern blot analysis of 293, 911 and PER celllines. Cellular DNA was extracted, HindIII digested, electrophoresed andtransferred to Hybond N+membranes (Amersham).

FIG. 9 illustrates the transfection efficiency of PER.C3, PER.C5,PER.C6™ and 911 cells.

FIG. 10 illustrates construction of adenovirus vector, pMLPI.TK.pMLPI.TK designed to have no sequence overlap with the packagingconstruct pIG.E1A.E1B.

FIGS. 11A and 11B illustrate new adenovirus packaging constructs whichdo not have sequence overlap with new adenovirus vectors.

FIG. 12 illustrate the generation of recombinant adenovirus,IG.Ad.MLPI.TK.

FIG. 13 illustrates the adenovirus double-stranded DNA genome indicatingthe approximate locations of E1, E2, E3, E4, and L regions.

FIG. 14 illustrates the adenovirus genome is shown in the top left withthe origins of replication located within the left and right ITRs at thegenome ends.

FIG. 15 illustrates a potential hairpin conformation of asingle-stranded DNA molecule that contains the HP/asp sequence.

FIG. 16 illustrates a diagram of pICLhac.

FIG. 17 illustrates a diagram of pICLhaw.

FIG. 18 illustrates a schematic representation of pICLI.

FIG. 19 is a diagram of pICL.

The so-called “minimal” adenovirus vectors according to the presentinvention retain at least a portion of the viral genome that is requiredfor encapsidation of the genome into virus particles (the encapsidationsignal), as well as at least one copy of at least a functional part or aderivative of the ITR, that is DNA sequences derived from the termini ofthe linear adenovirus genome. The vectors according to the presentinvention will also contain a transgene linked to a promoter sequence togovern expression of the transgene. Packaging of the so-called minimaladenovirus vector can be achieved by co-infection with a helper virusor, alternatively, with a packaging deficient replicating helper systemas described below.

Adenovirus-derived DNA fragments that can replicate in suitable celllines and that may serve as a packaging deficient replicating helpersystem are generated as follows. These DNA fragments retain at least aportion of the transcribed region of the “late” transcription unit ofthe adenovirus genome and carry deletions in at least a portion of theE1 region and deletions in at least a portion of the encapsidationsignal. In addition, these DNA fragments contain at least one copy of anITR. At one terminus of the transfected DNA molecule an ITR is located.The other end may contain an ITR, or alternatively, a DNA sequence thatis complementary to a portion of the same strand of the DNA moleculeother than the ITR. If, in the latter case, the two complementarysequences anneal, the free 3′-hydroxyl group of the 3′ terminalnucleotide of the hairpin-structure can serve as a primer for DNAsynthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion into a double-stranded form of at least aportion of the DNA molecule. Further replication initiating at the ITRwill result in a linear double-stranded DNA molecule, that is flanked bytwo ITR's, and is larger than the original transfected DNA molecule (seeFIG. 13). This molecule can replicate itself in the transfected cell byvirtue of the adenovirus proteins encoded by the DNA molecule and theadenoviral and cellular proteins encoded by genes in the host-cellgenome. This DNA molecule can not be encapsidated due to its large size(greater than 39,000 base pairs) or due to the absence of a functionalencapsidation signal. This DNA molecule is intended to serve as a helperfor the production of defective adenovirus vectors in suitable celllines.

The invention also comprises a method for amplifying linear DNAfragments of variable size in suitable mammalian cells. These DNAfragments contain at least one copy of the ITR at one of the termini ofthe fragment. The other end may contain an ITR, or alternatively, a DNAsequence that is complementary to a portion of the same strand of theDNA molecule other than the ITR. If, in the latter case, the twocomplementary sequences anneal, the free 3′-hydroxyl group of the 3′terminal nucleotide of the hairpin-structure can serve as a primer forDNA synthesis by cellular and/or adenovirus-encoded DNA polymerases,resulting in conversion of the displaced stand into a double strandedform of at least a portion of the DNA molecule. Further replicationinitiating at the ITR will result in a linear double-stranded DNAmolecule, that is flanked by two ITR's, which is larger than theoriginal transfected DNA molecule. A DNA molecule that contains ITRsequences at both ends can replicate itself in transfected cells byvirtue of the presence of at least the adenovirus E2 proteins (viz. theDNA-binding protein (“DBP”), the adenovirus DNA polymerase (“Ad-pol”),and the pre-terminal protein (“pTP”). The required proteins may beexpressed from adenovirus genes on the DNA molecule itself, fromadenovirus E2 genes integrated in the host-cell genome, or from areplicating helper fragment as described above.

Several groups have shown that the presence of ITR sequences at the endof DNA molecules are sufficient to generate adenovirus minichromosomesthat can replicate, if the adenovirus-proteins required for replicationare provided in trans, for example, by infection with a helpervirus (Huet al., 1992; Wang and Pearson, 1985; Hay et al., 1984). Hu et al.(1992) observed the presence and replication or symmetrical adenovirusminichromosome-dimers after transfection of plasmids containing a singleITR. The authors were able to demonstrate that these dimericminichromosomes arise after tail-to-tail ligation of the single ITR DNAmolecules. In DNA extracted from defective adenovirus type 2 particles,dimeric molecules of various sizes have also been observed usingelectron-microscopy (Daniell, 1976). It was suggested that theincomplete genomes were formed by illegitimate recombination betweendifferent molecules and that variations in the position of the sequenceat which the illegitimate base pairing occurred were responsible for theheterogeneous nature of the incomplete genomes. Based on this mechanismit was speculated that, in theory, defective molecules with a totallength of up to two times the normal genome could be generated. Suchmolecules could contain duplicated sequences from either end of thegenome. However, no DNA molecules larger than the full-length virus werefound packaged in the defective particles (Daniell, 1976). This can beexplained by the size-limitations that apply to the packaging. Inaddition, it was observed that in the virus particles DNA-molecules witha duplicated left-end predominated over those containing the right-endterminus (Daniell, 1976). This is fully explained by the presence of theencapsidation signal near that left-end of the genome (Gräble andHearing, 1990; Gräble and Hearing, 1992; Hearing et al., 1987).

The major problems associated with the current adenovirus-derivedvectors are:

1) The strong immunogenicity of the virus particle.

2) The expression of adenovirus genes that reside in the adenoviralvectors, resulting in a Cytotoxic T-cell response against the transducedcells.

3) The low amount of heterologous sequences that can be accommodated inthe current vectors (Up to maximally approx. 8000 base pairs (“bp”) ofheterologous DNA).

The strong immunogenicity of the adenovirus particle results in animmunological response of the host, even after a single administrationof the adenoviral vector. As a result of the development of neutralizingantibodies, a subsequent administration of the virus will be lesseffective or even completely ineffective. However, a prolonged orpersistent expression of the transferred genes will reduce the number ofadministrations required and may bypass the problem.

With regard to problem 2), experiments performed by Wilson andcollaborators have demonstrated that after adenovirus-mediated genetransfer into immunocompetent animals, the expression of the transgenegradually decreases and disappears approximately 2-4 weekspost-infection (Yang et al. 1994a; Yang et al., 1994b). This is causedby the development of a Cytotoxic T-Cell (“CTL”) response against thetransduced cells. The CTLs were directed against adenovirus proteinsexpressed by the viral vectors. In the transduced cells synthesis of theadenovirus DNA-binding protein (the E2A-gene product), penton and fiberproteins (late-gene products) could be established. These adenovirusproteins, encoded by the viral vector, were expressed despite deletionof the E1 region. This demonstrates that deletion of the E1 region isnot sufficient to completely prevent expression of the viral genes(Engelhardt et al., 1994a).

With regard to problem 3), studies by Graham and collaborators havedemonstrated that adenoviruses are capable of encapsidating DNA of up to105% of the normal genome size (Bett et al., 1993). Larger genomes tendto be instable resulting in loss of DNA sequences during propagation ofthe virus. Combining deletions in the E1 and E3 regions of the virualgenomes increases the maximum size of the foreign DNA that can beencapsidated to approximately 8.3 kb. In addition, some sequences of theE4 region appear to be dispensable for virus growth (adding another 1.8kb to the maximum encapsidation capacity). Also the E2A region can bedeleted from the vector, when the E2A gene product is provided in transin the encapsidation cell line, adding another 1.6 kb. It is, however,unlikely that the maximum capacity of foreign DNA can be significantlyincreased further than 12 kb.

We developed a new strategy for the generation and production ofhelper-free-stocks of recombinant adenovirus vectors that canaccommodate up to 38 kb of foreign DNA. Only two functional ITRsequences and sequences that can function as an encapsidation signalneed to be part of the vector genome. Such vectors are called “minimaladenovectors.” The helper functions for the minimal adenovectors areprovided in trans by encapsidation defective-replication competent DNAmolecules that contain all the viral genes encoding the required geneproducts, with the exception of those genes that are present in thehost-cell genome, or genes that reside in the vector genome.

The applications of the disclosed inventions are outlined below and willbe illustrated in the experimental part, which is only intended for thatpurpose, and should not be used to reduce the scope of the presentinvention as understood by the person skilled in the art.

Use of the IG Packaging Constructs Diploid Cells

The constructs, in particular pIG.E1A.E 1B, will be used to transfectdiploid human cells, such as HER, HEK, and Human Embryonic Lung cells(“HEL”). Transfected cells will be selected for transformed phenotype(focus formation) and tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Such celllines will be used for the generation and (large-scale) production ofE1-deleted recombinant adenoviruses. Such cells, infected withrecombinant adenovirus are also intended to be used in vivo as a localproducer of recombinant adenovirus, for example, for the treatment ofsolid tumors.

911 cells are used for the titration, generation and production ofrecombinant adenovirus vectors (Fallaux et al., 1996).

HER cells transfected with pIG.E1A.E1B have resulted in 7 independentclones (called PER cells). These clones are used for the production ofE1-deleted (including non-overlapping adenovirus vectors) or E1defective recombinant adenovirus vectors, and provide the basis forintroduction of, for example, E2B or E2A constructs (e.g., ts125E2A, seebelow), E4 etc., that will allow propagation of adenovirus vectors thathave mutations in, for example, E2A or E4.

In addition, diploid cells of other species that are permissive forhuman adenovirus, such as the cotton rat (Sigmodon hispidus) (Pacini etal., 1984), Syrian hamster (Morin et al., 1987) or chimpanzee (Levreroet al., 1991), will be immortalized with these constructs. Such cells,infected with recombinant adenovirus are also intended to be used invivo for the local production of recombinant adenovirus, for example,for the treatment of solid tumors.

Established Cells

The constructs, in particular pIG.E1A.NEO, can be used to transfectestablished cells, for example, A549 (human bronchial carcinoma), KB(oral carcinoma), MRC-5 (human diploid lung cell line) or GLC cell lines(small cell lung cancer) (de Leij et al., 1985; Postmus et al., 1988)and selected for NEO resistance. Individual colonies of resistant cellsare isolated and tested for their capacity to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK. Whenpropagation of E1-deleted viruses on E1A containing cells is possible,such cells can be used for the generation and production of E1-deletedrecombinant adenovirus. They can also be used for the propagation of E1Adeleted/E1B retained recombinant adenovirus

Established cells can also be co-transfected with pIG.E1A.E1B andpIG.NEO (or another NEO containing expression vector). Clones resistantto G418 are tested for their ability to support propagation ofE1-deleted recombinant adenovirus, such as IG.Ad.MLPI.TK and used forthe generation and production of E1 deleted recombinant adenovirus andwill be applied in vivo for local production of recombinant virus, asdescribed for the diploid cells (see previous discussion). All celllines, including transformed diploid cell lines or NEO-resistantestablished lines, can be used as the basis for the generation of ‘nextgeneration’ packaging cells lines, that support propagation ofE1-defective recombinant adenoviruses, that also carry deletions inother genes, such as E2A and E4. Moreover, they will provide the basisfor the generation of minimal adenovirus vectors as disclosed herein.

E2 Expressing Cell Lines

Packaging cells expressing E2A sequences are and will be used for thegeneration and large scale production of E2A-deleted recombinantadenovirus.

The newly generated human adenovirus packaging cell lines or cell linesderived from species permissive for human adenovirus (E2A orts125E2A:E1A+E2A;E1A+E1B+E2A, E1A−E2A/ts125;E1A+E1B−E2A/ts125) ornon-permissive cell lines such as monkey cells (hrE2A orhr+ts125E2A;E1A+hrE2A;E1A+E1B+hrE2A;E1A+hrE2A/ts125;E1A−E1B+E1A−E1B+hrE2A/ts125)are and will be used for the generation and large scale production ofE2A deleted recombinant adenovirus vectors. In addition, they will beapplied in vivo for local production of recombinant virus, as describedfor the diploid cells (see previous discussion).

Novel Adenovirus Vectors

The newly developed adenovirus vectors harboring an E1 deletion of nt.459-3510 will be used for gene transfer purposes. These vectors are alsothe basis for the development of further deleted adenovirus vectors thatare mutated for, for example, E2A, E2B or E4. Such vectors will begenerated, for example, on the newly developed packaging cell linesdescribed above.

Minimal Adenovirus Packaging System

We disclose adenovirus packaging constructs (to be used for thepackaging of minimal adenovirus vectors) which have the followingcharacteristics:

1) the packaging construct replicates;

2) the packaging construct can not be packaged because the packagingsignal is deleted;

3) the packaging construct contains an internal hairpin-forming sequence(see FIG. 15);

4) because of the internal hairpin structure, the packaging construct isduplicated, that is, the DNA of the packaging construct becomes twice aslong as it was before transfection into the packaging cell (in oursample it duplicates from 35 kb to 70 kb). This duplication alsoprevents packaging. Note that this duplicated DNA molecule has ITR's atboth termini (see, e.g., FIG. 13);

5) this duplicated packaging molecule is able to replicate like a‘normal adenovirus’ DNA molecule;

6) the duplication of the genome is a prerequisite for the production ofsufficient levels of adenovirus proteins, required to package theminimal adenovirus vector; and

7) the packaging construct has no overlapping sequences with the minimalvector or cellular sequences that may lead to generation of RCA byhomologous recombination.

This packaging system is used to produce minimal adenovirus vectors. Theadvantages of minimal adenovirus vectors, for example, for gene therapyof vaccination purposes, are well known (accommodation of up to 38 kb;gutting of potentially toxic and immunogenic adenovirus genes).

Adenovirus vectors containing mutations in essential genes (includingminimal adenovirus vectors) can also be propagated using this system.

Use of Intracellular E2 Expressing Vectors

Minimal adenovirus vectors are generated using the helper functionsprovided in trans by packaging-deficient replicating helper molecules.The adenovirus-derived ITR sequences serve as origins of DNA replicationin the presence of at least the E2-gene products. When the E2 geneproducts are expressed from genes in the vector genome (the gene(s) mustbe driven by an E1-independent promoter), the vector genome canreplicate in the target cells. This will allow a significantly increasednumber of template molecules in the target cells, and, as a result, anincreased expression of the genes of interest encoded by the vector.This is of particular interest for application of gene therapy in cancertreatment.

Applications of Intracellular Amplification of Linear DNA Fragments

A similar approach could also be taken if amplification of linear DNAfragments is desired. DNA fragments of known or unknown sequence couldbe amplified in cells containing the E2-gene products if at least oneITR sequence is located near or at its terminus. There are no apparentconstraints on the size of the fragment. Even fragments much larger thanthe adenovirus genome (36 kb) should be amplified using this approach.It is thus possible to clone large fragments in mammalian cells withouteither shuttling the fragment into bacteria (such as E. coli) or use thepolymerase chain reaction (“PCR”). At the end stage of an productiveadenovirus infection a single cell can contain over 100,000 copies ofthe viral genome. In the optimal situation, the Linear DNA fragments canbe amplified to similar levels. Thus, one should be able to extract morethan 5 μg of DNA fragment per 10 million cells (for a 35-kbp fragment).This system can be used to express heterologous proteins equivalent tothe Simian Virus 40-based COS-cell system) for research or fortherapeutic purposes. In addition, the system can be used to identifygenes in large fragments of DNA. Random DNA fragments may be amplified(after addition of ITRs) and expressed during intracellularamplification. Election or selection of those cells with the desiredphenotype can be used to enrich the fragment of interest and to isolatethe gene.

EXPERIMENTAL

Generation of Cell Lines able to Transcomplement E1 DefectiveRecombinant Adenovirus Vectors

911 Cell Line

We have generated a cell line that harbors E1 sequences of adenovirustype 5 (“Ad5”), able to trans-complement E1 deleted recombinantadenovirus (Fallaux et al., 1996). This cell line was obtained bytransfection of human diploid human embryonic retinoblasts (“HER”) withpAd5XhoIC, that contains nt. 80-5788 of Ad5; one of the resultingtransformants was designated 911. This cell line has been shown to bevery useful in the propagation of E1 defective recombinant adenovirus.It was found to be superior to 293 cells. Unlike 293 cells, 911 cellslack a fully transformed phenotype, which most likely is the cause ofits better performance as adenovirus packaging line:

1) plaque assays can be performed faster (4-5 days instead of 8-14 dayson 293),

2) monolayers of 911 cells survive better under agar overlay as requiredfor plaque assays, and

3) higher amplification of E1-deleted vectors is obtained.

In addition, unlike 293 cells that were transfected with shearedadenoviral DNA, 911 cells were transfected using a defined construct.Transfection efficiencies of 911 cells are comparable to those of 293.

New Packaging Constructs. Source of Adenovirus Sequences

Adenovirus sequences are derived either from pAd5.SalB, containing nt.80-9460 of human adenovirus type 5 (Bernards et al., 1983) or fromwild-type Ad5DNA. pAd5.SalB was digested with SalI and XhoI and thelarge fragment was religated and this new clone was named pAd5.X/S. ThepTN construct (constructed by Dr. R. Vogels, IntroGene, Leiden, TheNetherlands) was used as a source for the human PGK promoter and the NEOgene.

Human PGK Promoter and NEO^(R) Gene

Transcription of E1A sequences in the new packaging constructs is drivenby the human PGK promoter (Michelson et al., 1983; Singer-Sam et al.,1984), derived from plasmid pTN (gift of R. Vogels), which uses pUC119(Vieira and Messing, 1987) as a backbone. This plasmid was also used asa source for NEO gene fused to the Hepatitis B Virus (“HBV”)poly-adenylation signal.

Fusion of PGK Promoter to E1 Genes

As shown in FIG. 1, in order to replace the E1 sequences of Ad5 (ITR,origin of replication and packaging signal) by heterologous sequences wehave amplified E1 sequences (nt.459 to nt. 960) of Ad5 by PCR, usingprimers Ea-1 (SEQ ID NO: 1) and Ea-2 (SEQ ID NO: 2) (see Table I). Theresulting PCR product was digested with ClaI and ligated into Bluescript(Stratagene), predigested with ClaI and EcoRV, resulting in constructpBS.PCRI.

TABLE I Primer Sequences. Name (SEQ ID NO) Sequence Function Primer Ea-1CGTGTAGTGT ATTTATACCC PCR amplification Ad5 (SEQ ID NO: 1) a G nt459 ->Primer Ea-2 TCGTCACTGG GTGGAAAGCC PCR amplification Ad5 (SEQ ID NO: 2) aA nt960 <- Primer Ea-3 TACCCGCCGT CCTAAAATGG nt1284-1304 of Ad5 (SEQ IDNO: 3) a C genome Primer Ea-5 TGGACTTGAG CTGTAAACGC nt1514-1533 of Ad5(SEQ ID NO: 4) a genome Primer Ep-2 GCCTCCATGG AGGTCAGATG nt1721-1702 ofAd5: (SEQ ID NO: 5) a T introduction of NcoI site Primer Eb-1 GCTTGAGCCCGAGACATGTC nt3269-3289 of Ad5 (SEQ ID NO: 6) a genome Primer Eb-2CCCCTCGAGC TCAATCTGTA nt3508-3496 of Ad5 (SEQ ID NO: 7) a TCTT genome:introduction of XhoI site Primer SV40-1 GGGGGATCCG AACTTGTTTAIntroduction BamHI (SEQ ID NO: 8) a TTGCAGC site (nt2182-2199 ofpMLP.TK) adaption of recombinant adenoviruses Primer SV40-2 GGGAGATCTAGACATGATAA Introduction BglII (SEQ ID NO: 9) a GATAC site (nt2312-2297of pMLP.TK) Primer Ad5-1 GGGAGATCTG TACTGAAATG Introduction of BglII(SEQ ID NO: 10) a TGTGGGC site (nt 2496-2514 of pMLP.TK) Primer Ad5-2GGAGGCTGCA GTCTCCAACG Rnt2779-2756 of (SEQ ID NO: 11) a GCGT PMLP.TKPrimer ITR1 GGGGGATCCT CAAATCGTCA nt35737-35757 of Ad5 (SEQ ID NO: 12) aCTTCCGT (introduction of BamBI site) Primer ITR2 GGGGTCTAGA CATCATCAATnt35935-35919 of Ad5 (SEQ ID NO: 13 a AATATAC (introduction of XbaIsite) PCR primer PCR/MLP1 GGCGAATTCG TCGACATCAT (Ad5 nt. 10-18) (SEQ IDNO: 14) b CAATAATATA CC PCT primer PCR/MLP2 GGCGAATTCG GTACCATCAT (Adsnt. 10-18) (SEQ ID NO: 15) b CAATAATATA CC PCT primer PCR/MLP3CTGTGTACAC CGGCGCA (Ad5 nt. 200-184) (SEQ ID NO: 16) b PCT primerHP/asp1 5′-GTACACTGAC CTAGTGCCGC (SEQ ID NO: 17) c    CCGGGCAAAGCCCGGGCGGC    ACTAGGTCAG PCT primer HP/asp2 5′-GTACCTGACC TAGTGCCGCC(SEQ ID NO: 18) c    CGGGCTTTGC CCGGGCGGCA    CTAGGTCAGT PCT primerHP/cla1 5′-GTACATTGAC CTAGTGCCGC (SEQ ID NO: 19) d    CCGGGCAAAGCCCGGGCGGC    ACTAGGTCAA TCGAT PCT primer HP/cla2 5′-GTACATCGATTGACCTAGTG (SEQ ID NO: 20) d    CCGCCCGGGC TTTGCCCGGG    CGGCACTAGGTCAAT a - Primers used for PCR amplification of DNA fragments used forgeneration of constructs described in this patent application. b - PCRprimers sets to be used to create the SalI and Asp7l8 sites juxtaposedto the ITR sequences. c - Synthetic oligonucleotide pair used togenerate a synthetic hairpin, recreates an Asp718 site at one of thetermini if inserted in Asp718 site. d - Synthetic oligonucleotide pairused to generate a synthetic hairpin, contains the ClaI recognition siteto be used for hairpin formation.

Vector pTN was digested with restriction enzymes EcoRI (partially) andScaI. and the DNA fragment containing the PGK promoter sequences wasligated into PBS.PCRI digested with ScaI and EcoRI. The resultingconstruct PBS.PGK.PCRI contains the human PGK promoter operativelylinked to Ad5 E1 sequences from nt.459 to nt. 916.

Construction of pIG.E1A.E1B.X

As shown in FIG. 2, pIG.E1A.E1B.X was made by replacing the ScaI-BspEIfragment of pAT-X/S by the corresponding fragment from PBS.PGK.PCRI(containing the PGK promoter linked to E1A sequences). pIG.E1A.E1B.Xcontains the E1A and E1B coding sequences under the direction of the PGKpromoter. As Ad5 sequences from nt.459 to nt. 5788 are present in thisconstruct, also pIX protein of adenovirus is encoded by this plasmid.

Construction of pIG.E1A.NEO

As shown in FIG. 3A, in order to introduce the complete E1B promoter andto fuse this promoter in such a way that the AUG codon of E1B21 kDexactly functions as the AUG codon of NEO^(R), we amplified the E1Bpromoter using primers Ea-3 (SEQ ID NO: 3) and Ep-2(SEQ ID NO: 5), whereprimer Ep-2 introduces an NcoI site in the PCR fragment. The resultingPCR fragment, named PCRII, was digested with HpaI and NcoI and ligatedinto pAT-X/S, which was predigested with HpaI and with NcoI. Theresulting plasmid was designated pAT-X/S-PCR2. The NcoI-StuI fragment ofpTN, containing the NEO gene and part of the HBV poly-adenylationsignal, was cloned into pAT-X/S-PCR2 (digested with NcoI and NruI). Theresulting construct: pAT-PCR2-NEO.

As shown in FIG. 3B, the poly-adenylation signal was completed byreplacing the ScaI-SalI fragment of pAT-PCR2-NEO by the correspondingfragment of pTN (resulting in pAT.PCR2.NEO.p(A)). The ScaI-XbaI ofpAT.PCR2.NEO.p (A) was replaced by the corresponding fragment ofpIG.E1A.E1B-X, containing the PGK promoter linked to E1A genes. Theresulting construct was named pIG.E1A.NEO, and thus contains Ad5 E1sequences (nt.459 to nt 1713) under the control of the human PGKpromoter.

Construction of pIG.E1A.E1B

As shown in FIG. 4, pIG.E1A.E1B was made by amplifying the sequencesencoding the N-terminal amino acids of E1B 55kd using primers Eb-1 (SEQID NO: 6) and Eb-2 (SEQ ID NO: 7) (introduces a XhoI site). Theresulting PCR fragment was digested with BglII and cloned intoBglII/NruI of pAT-X/S, thereby obtaining pAT-PCR3.

pIG.E1A.E1B was constructed by introducing the HBV poly(A) sequences ofpIG.E1A.NEO downstream of E1B sequences of pAT-PCR3 by exchange ofXbaI-SalI fragment of pIg.E1A.NEO and the XbaI XhoI fragment ofpAT.PCR3.

pIG.E1A.E1B contains nt. 459 to nt. 3510 of Ad5, that encode the E1A andE1B proteins. The E1B sequences are terminated at the splice acceptor atnt.35 11. No pIX sequences are present in this construct.

Construction of pIG.NEO

As shown in FIG. 5, pIG.NEO was generated by cloning the HpaI-ScaIfragment of pIG.E1A.NEO, containing the NEO gene under the control ofthe Ad.5E1B promoter, into pBS digested with EcoRV and ScaI.

This construct is of use when established cells are transfected withE1A.E1B constructs and NEO selection is required. Because NEO expressionis directed by the E1B promoter, NEO resistant cells are expected toco-express E1A, which also is advantageous for maintaining high levelsof expression of E1A during long-term culture of the cells.

Testing of Constructs

The integrity of the constructs pIG.E1A.NEO,pIG.E1A.E1B.X andpIG.E1A.E1B was assessed by restriction enzyme mapping; furthermore,parts of the constructs that were obtained by PCR analysis wereconfirmed by sequence analysis. No changes in the nucleotide sequencewere found.

The constructs were transfected into primary Baby Rat Kidney (“BRK”)cells and tested for their ability to immortalize (pIG.E1A.NEC) or fullytransform (pAd5.XhoIC,pIG.E1A.E1B.X and pIG.E1A.E1B) these cells.

Kidneys of 6-day old WAG-Rij rats were isolated, homogenized andtrypsinized. Subconfluent dishes (diameter 5 cm) of the BRK cellcultures were transfected with 1 or 5 μg of pIG.NEO, pIG.E1A.NEO,pIG.E1A.E1B, pIG.E1A.E1B.X, pAd5XhoIC, or with pIG.E1A.NEO together withPDC26 (Van der Elsen et al., 1983), carrying the Ad5.E1B gene undercontrol of the SV4O early promoter. Three weeks post-transfection, whenfoci were visible, the dishes were fixed, Giemsa stained and the focicounted.

An overview of the generated adenovirus packaging constructs, and theirability to transform BRK, is presented in FIG. 6. The results indicatethat the constructs pIG.E1A.E1B and pIG.E1A.E1B.X are able to transformBRK cells in a dose-dependent manner. The efficiency of transformationis similar for both constructs and is comparable to what was found withthe construct that was used to make 911 cells, namely pAd5. XhoIC.

As expected, pIG.E1A.NEO was hardly able to immortalize BRK. However,co-transfection of an E1B expression construct (PDC26) did result in asignificant increase of the number of transformants (18 versus 1),indicating that E1A encoded by pIG.E1A.NEO is functional. We conclude,therefore, that the newly generated packaging constructs are suited forthe generation of new adenovirus packaging lines.

Generation of Cell Lines with New Packaging Constructs Cell Lines andCell Culture

Human A549 bronchial carcinoma cells (Shapiro et al., 1978), humanembryonic retinoblasts (“HER”), Ad5-E1-transformed human embryonickidney (“HEK”) cells (293; Graham et al., 1977) cells andAd5-transformed HER cells (911; Fallaux et al., 1996)) and PER cellswere grown in Dulbecco's Modified Eagle Medium (“DMEM”) supplementedwith 10% Fetal Calf Serum (“FCS”) and antibiotics in a 5% C02 atmosphereat 37° C. Cell culture media, reagents and sera were purchased fromGibco Laboratories (Grand Island, N.Y.). Culture plastics were purchasedfrom Greiner (Nürtingen, Germany) and Corning (Corning, N.Y.).

Viruses and Virus Techniques

The construction of adenoviral vectors IG.Ad.MLP.nls.lacZ,IG.Ad.MLP.luc, IG.Ad.MLP.TK and IG.Ad.CMV.TK is described in detail inpatent application EP 95202213. The recombinant adenoviral vectorIG.Ad.MLP.nls.lacZ contains the E. coli lacZ gene, encodingβ-galactosidase, under control of the Ad2 major late promoter (“MLP”).IG.Ad.MLP.luc contains the firefly luciferase gene driven by the Ad2MLP. Adenoviral vectors IG.Ad.MLP.TK and IG.Ad.CMV.TR contain the HerpesSimplex Virus thymidine kinase (“TK”) gene under the control of the Ad2MLP and the Cytomegalovirus (“CMV”) enhancer/promoter, respectively.

Transfections

All transfections were performed by calcium-phosphate precipitation DNA(Graham and Van der Eb, 1973) with the GIBCO Calcium PhosphateTransfection System (GIBCO BRL Life Technologies Inc., Gaithersburg,Md., USA), according to the manufacturers protocol.

Western Blotting

Subconfluent cultures of exponentially growing 293,911 andAd5-E1-transformed A549 and PER cells were washed with PBS and scrapedin Fos-RIPA buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP4O,01% sodiumdodecyl sulphate (“SDS”), 1% NA-DOC, 0.5 mM phenyl methyl sulphonylfluoride (“PMSF”), 0.5 mM trypsin inhibitor, 50 mM NaF and 1 mM sodiumvanadate). After 10 minutes at room temperature, lysates were cleared bycentrifugation. Protein concentrations were measured with the Bioradprotein assay kit, and 25 μg total cellular protein was loaded on a12.5% SDS-PAA gel. After electrophoresis, proteins were transferred tonitrocellulose (1 hour at 300 mA). Prestained standards (Sigma, USA)were run in parallel. Filters were blocked with 1% bovine serum albumin(“BSA”) in TBST (10 mM Tris, pH 8.15 mM NaCl, and 0.05% TWEEN™-20) for 1hour. First antibodies were the mouse monoclonal anti-Ad5-E1B-55-kDAantibody A1C6 (Zantema et al, unpublished), the rat monoclonalanti-Ad5-E1B-22 1-kDa antibody ClGll (Zantema et al, 1985). The secondantibody was a horseradish peroxidase-labeled goat anti-mouse antibody(Promega). Signals were visualized by enhanced chemoluminescence(Amersham Corp, UK).

Southern Blot Analysis

High molecular weight DNA was isolated and 10 μg was digested tocompletion and fractionated on a 0.7% agarose gel. Southern blottransfer to Hybond N+ (Amersham, UK) was performed with a 0.4 M NAOH,0.6 M NaCl transfer solution (Church and Gilbert, 1984). Hybridizationwas performed with a 2463-nt SspI-HindIII fragment from pAd5.SalB(Bernards et al., 1983). This fragment consists of Ad5 bp. 342-2805. Thefragment was radiolabeled with α-^(32p)-dCTP with the use of randomhexanucleotide primers and Klenow DNA polymerase. The southern blotswere exposed to a Kodak XAR-5 film at −80° C. and to a Phospho-Imagerscreen which was analyzed by B&L systems Molecular Dynamics software.

A549

Ad5-E1-transformed A549 human bronchial carcinoma cell lines weregenerated by transfection with pIG.E1A.NEO and selection for G418resistance. Thirty-one G418 resistant clones were established.Co-transfection of pIG.E1A.E1B with pIG.NEO yielded seven G418 resistantcell lines.

PER

Ad5-E1-transformed HER cells were generated by transfection of primaryHER cells with plasmid pIG.E1A.E1B. Transformed cell lines wereestablished from well-separated foci. We were able to establish sevenclonal cell lines which we called PER.C1, PER.C3, PER.C4, PER.C5,PER.C6, PER.C8, and PER.C9. One of the PER clones, namely PER.C6, hasbeen deposited under the Budapest Treaty under number ECACC 96022940with the Centre for Applied Microbiology and Research of Porton Down, UKon Feb. 29, 1996. In addition, PER.C6 is commercially available fromIngroGene, B.V., Leiden, NL.

Expression of Ad5 E1A and E1B Genes in Transformed A549 and PER Cells

Expression of the Ad5 E1A and the 55-kDa and 21 kDa E1B proteins in theestablished A549 and PER cells was studied by means of Western blotting,with the use of monoclonal antibodies (“Mab”). Mab M73 recognizes theE1A products, whereas Mabs AIC6 and C1G11 are directed against the55-kDa and 21 kDa E1B proteins, respectively.

The antibodies did not recognize proteins in extracts from the parentalA549 or the primary HER cells (data not shown). None of the A549 clonesthat were generated by co-transfection of pIG.NEO and pIG.E1A.E1Bexpressed detectable levels of E1A or E1B proteins (not shown). Some ofthe A549 clones that were generated by transfection with pIG.E1A.NEOexpressed the Ad5 E1A proteins (FIG.7), but the levels were much lowerthan those detected in protein lysates from 293 cells. The steady stateE1A levels detected in protein extracts from PER cells were much higherthan those detected in extracts from A549-derived cells. All PER celllines expressed similar levels of E1A proteins (FIG. 7). The expressionof the E1B proteins, particularly in the case of E1B 55 kDa, was morevariable. Compared to 911 and 293, the majority of the PER clonesexpress high levels of E1B 55 kDa and 21 kDa. The steady state level ofE1B 21 kDa was the highest in PER. C3. None of the PER clones lostexpression of the Ad5 E1 genes upon serial passage of the cells (notshown). We found that the level of E1 expression in PER cells remainedstable for at least 100 population doublings. We decided to characterizethe PER clones in more detail.

Southern Analysis of PER Clones

To study the arrangement of the Ad5-E1 encoding sequences in the PERclones we performed Southern analyses. Cellular DNA was extracted fromall PER clones, and from 293 and 911 cells. The DNA was digested withHindIII, which cuts once in the Ad5 E1 region. Southern hybridization onHindIII-digested DNA, using a radiolabeled Ad5-E1-specific proberevealed the presence of several integrated copies of pIG.E1A.E1B in thegenome of the PER clones. FIG. 8 shows the distribution pattern of E1sequences in the high molecular weight DNA of the different PER celllines. The copies are concentrated in a single band, which suggests thatthey are integrated as tandem repeats. In the case of PER.C3, C5, C6 andC9 we found additional hybridizing bands of low molecular weight thatindicate the presence of truncated copies of pIG.E1A.E1B. The number ofcopies was determined with the use of a Phospho-Imager. We estimatedthat PER.C1, C3, C4, C5, C6, C8 and C9 contain 2, 88, 5,4, 5, 5 and 3copies of the Ad5 E1 coding region, respectively, and that 911 and 293cells contain 1 and 4 copies of the Ad5 E1 sequences, respectively.

Transfection Efficiency

Recombinant adenovectors are generated by co-transfection of adaptorplasmids and the large ClaI fragment of Ad5 into 293 cells (see EuropeanPatent Office (“EPO”) application EP 95202213). The recombinant virusDNA is formed by homologous recombination between the homologous viralsequences that are present in the plasmid and the adenovirus DNA. Theefficacy of this method, as well as that of alternative strategies, ishighly dependent on the transfectability of the helper cells. Therefore,we compared the transfection efficiencies of some of the PER clones with911 cells, using the E. coli β-galactosidase-encoding lacZ gene as areporter (FIG. 9).

Production of Recombinant Adenovirus

Yields of recombinant adenovirus obtained after inoculation of293, 911,PER.C3, PER.C5 and PER.C6 with different adenovirus vectors arepresented in Table II. The results indicate that the yields obtained onPER cells are at least as high as those obtained on the existing celllines. In addition, the yields of the novel adenovirus vectorIG.Ad.MLPI.TK are similar or higher than the yields obtained for theother viral vectors on all cell lines tested.

TABLE II IG.Ad. Pro- Passage CMV. IG.Ad. IG.Ad. ducer Cell number lacZCMV.TK MLPI.TK d1313 Mean 293 6.0 5.8 24 34 17.5 911 8 14 34 180 59.5PER.C3 17 8 11 44 40 25.8 PER.C5 15 6 17 36 200 64.7 PER.C6 36 10 22 58320 102 Yields × 10⁻⁸ pfu/T175 flask.

Table II. Yields of different recombinant adenoviruses obtained afterinoculation of adenovirus E1 packaging cell lines 293, 911, PER.C3,PER.C5 and PER.C6. The yields are the mean of two different experiments.IG.Ad.CMV.lacZ and IG.Ad.CMV.TK are described in patent application EP95 20 2213. The construction of IG.Ad.MLPI.TK is described in thispatent application. Yields of virus per T80 flask were determined byplaque assay on 911 cells, as described [Fallaux, 1996 #1493]

Generation of New Adenovirus Vectors

The used recombinant adenovirus vectors (see EPO patent application no.EP 95202213) are deleted for E1 sequences from 459 to nt. 3328.

As construct pE1A.E1B contains Ad5 sequences 459 to not 3510 there is asequence overlap of 183 nt. between E1B sequences in the packagingconstruct pIG.E1A.E1B and recombinant adenoviruses, such as, forexample, IG.Ad.MLP.TK. The overlapping sequences were deleted from thenew adenovirus vectors. In addition, non-coding sequences derived fromlacZ, that are present in the original constructs, were deleted as well.This was achieved (see FIG. 10) by PCR amplification of the SV4O poly(A)sequences from pMLP.TK using primers SV40-1 (SEQ ID NO: 8) (introduces aBamHI site) and SV4O-2 (SEQ ID NO: 9) (introduces a BglII site). Inaddition, Ad5 sequences present in this construct were amplified from nt2496 (Ad5-1 (SEQ ID NO: 10), introduces a BglII site) to nt. 2779 (Ad5-2(SEQ ID NO: 11)). Both PCR fragments were digested with BglII and wereligated. The ligation product was PCR amplified using primers SV40-1 andAd5-2. The PCR product obtained was cut with BamHI and AflII and wasligated into pMLP.TK predigested with the same enzymes. The resultingconstruct, named pMLPI.TK, contains a deletion in adenovirus E1sequences from nt 459 to nt. 3510.

Packaging System

The combination of the new packaging construct pIG.E1A.E1B and therecombinant adenovirus pMLPI.TK, which do not have any sequence overlap,are presented in FIGS. 11A and 11B. In these figures, the originalsituation is also presented, with the sequence overlap indicated.

The absence of overlapping sequences between pIG.E1A.E1B and pMLPI.TK(FIG. 11A) excludes the possibility of homologous recombination betweenpackaging construct and recombinant virus, and is therefore asignificant improvement for production of recombinant adenovirus ascompared to the original situation.

In FIG. 11B the situation is depicted for pIG.E1A.NEO and IG.Ad.MLPI.TK.pIG.E1A.NEO, when transfected into established cells, is expected to besufficient to support propagation of E1-deleted recombinant adenovirus.This combination does not have any sequence overlap, preventinggeneration of RCA by homologous recombination. In addition, thisconvenient packaging system allows the propagation of recombinantadenoviruses that are deleted just for E1A sequences and not for E1Bsequences. Recombinant adenoviruses expressing E1B in the absence of E1Aare attractive, as the E1B protein, in particular E1B19kD, is able toprevent infected human cells from lysis by Tumor Necrosis Factor (“TNF”)(Gooding et al., 1991).

Generation of Recombinant Adenovirus Derived from pMLPI.TK

Recombinant adenovirus was generated by co-transfection of 293 cellswith SalI linearized pMLPI.TK DNA and ClaI linearized Ad5 wt DNA. Theprocedure is schematically represented in FIG. 12.

Outline of the Strategy to Generate Packaging Systems for MinimalAdenovirus Vector

Name convention of the plasmids used:

p plasmid

I ITR (Adenovirus Inverted Terminal Repeat)

C CMV Enhancer/Promoter Combination

L Firefly Luciferase Coding Sequence hac,haw Potential hairpin that canbe formed after digestion with restriction endonuclease Asp718 in itscorrect and in the reverse orientation, respectively (FIG. 15 (SEQ IDNO: 22)).

For example, pICLhaw is a plasmid that contains the adenovirus ITRfollowed by the CMV-driven luciferase gene and the Asp718 hairpin in thereverse (non-functional) orientation.

Experiment Series 1

The following demonstrates the competence of a synthetic DNA sequencethat is capable of forming a hairpin-structure to serve as a primer forreverse strand synthesis for the generation of double-stranded DNAmolecules in cells that contain and express adenovirus genes.

Plasmids pICLhac, pICLhaw, pICLI and pICL were generated using standardtechniques. The schematic representation of these plasmids is shown inFIGS. 16-19.

Plasmid pICL is derived from the following plasmids:

nt.1-457 pMLP10 (Levrero et al., 1991)

nt.458-1218 pCMVβ (Clontech, EMBL Bank No. U02451)

nt.1219-3016 pMLP.luc (IntroGene, Leiden, NL, unpublished)

nt.3017-5620 pBLCAT5 (Stein and Whelan, 1989)

The plasmid has been constructed as follows:

The tet gene ofplasmid pMLP10 has been inactivated by deletion of theBamHI-SalI fragment, to generate pMLP10ΔSB. Using primer set PCR/MLP1(SEQ ID NO: 14) and PCRIMLP3 (SEQ ID NO: 16) a 210 bp fragmentcontaining the Ad5-ITR, flanked by a synthetic SalI restriction site wasamplified using pMLP10 DNA as the template. The PCR product was digestedwith the enzymes EcoRI and SgrAI to generate a 196 bp. fragment. PlasmidpMLP10ΔSB was digested with EcoRI and SgrAI to remove the ITR. Thisfragment was replaced by the EcoRI-SgrAI-treated PCR fragment togenerate pMLP/SAL. Plasmid pCMV-Luc was digested with Pvull tocompletion and recirculated to remove the SV4O-derived poly-adenylationsignal and Ad5 sequences with exception of the Ad5 left-terminus. In theresulting plasmid, pCMV-lucΔAd, the Ad5 ITR was replaced by theSal-site-flanked ITR from plasmid pMLP/SAL by exchanging the XmnI-SacIIfragments. The resulting plasmid, pCMV-IucΔAdISAL, the Ad5 left terminusand the CMV-driven luciferase gene were isolated as an SalI-SmaIfragment and inserted in the SalI and HpaI digested plasmid pBLCATS, toform plasmid pICL. Plasmid pICL is represented in FIG. 19; its sequenceis presented in the sequence listing (SEQ ID NO: 21).

The plasmid pICL contains the following features: nt. 1-457 Ad5 leftterminus (Sequence 1-457 of human adenovirus type 5) nt. 458-969 Humancytomegalovirus enhancer and immediate early promoter (see Boshart etal., A Very Strong Enhancer is Located Upstream of an Immediate EarlyGene of Human Cytomegalovirus”, Cell 41, pp. 521- 530 (1985), herebyincorporated herein by reference) (from plasmid pCMVβ, Clontech, PaloAlto, USA) nt. 970-1204 SV40 19S exon and truncated 16/19S intron (fromplasmid pCMVβ) nt. 1218-2987 Firefly luciferase gene (from pMLP.luc) nt.3018-3131 SV40 tandem poly-adenylation signals from late trans- cript,derived from plasmid pBLCAT5) nt. 3132-5620 pUC12 backbone (derived fromplasmid pBLCAT5) nt. 4337-5191 β-lactamase gene (Amp-resistence gene,reverse orientation)

Plasmid pICLhac and pICLhaw

Plasmids pICLhac and pICLhaw were derived from plasmid pICL by digestionof the latter plasmid with the restriction enzyme Asp718. The linearizedplasmid was treated with Calf-Intestine Alkaline Phosphatase to removethe 51 phoshate groups. The partially complementary syntheticsingle-stranded oligonucleotide Hp/asp1 (SEQ ID NO: 17) and Hp/asp2 (SEQID NO: 18) were annealed and phosphorylated on their 5′ ends usingT4-polynucleotide kinase.

The phosporylated double-stranded oligomers were mixed with thedephosporylated pICL fragment and ligated. Clones containing a singlecopy of the synthetic oligonucleotide inserted into the plasmid wereisolated and characterized using restriction enzyme digests. Insertionof the oligonucleotide into the Asp718 site will at one junctionrecreate an Asp718 recognition site, whereas at the other junction therecognition site will be disrupted. The orientation and the integrity ofthe inserted oligonucleotide was verified in selected clones by sequenceanalyses. A clone containing the oligonucleotide in the correctorientation (the Asp718 site close to the 3205 EcoRI site) was denotedpICLhac. A clone with the oligonucleotide in the reverse orientation(the Asp718 site close to the SV40 derived poly signal) was designatedpICLhaw. Plasmids pICLhac and pICLhaw are represented in FIGS. 16 and17.

Plasmid pICLI was created from plasmid pICL by insertion of theSalI-SgrAI fragment from pICL, containing the Ad5-ITR into the Asp718site of pICL. The 194 bp SalI-SgrAI fragment was isolated from pICL, andthe cohesive ends were converted to blunt ends using E. coli DNApolymerase I (Klenow fragment) and dNTP's. The Asp718 cohesive ends wereconverted to blunt ends by treatment with mungbean nuclease. By ligationclones were generated that contain the ITR in the Asp718 site of plasmidpICL. A clone that contained the ITR fragment in the correct orientationwas designated pICLI (FIG. 18). Generation of adenovirus Ad-CMV-hcTK.Recombinant adenovirus was constructed according to the method describedin EPO Patent application 95202213. Two components are required togenerate a recombinant adenovirus. First an adaptor-plasmid containingthe left terminus of the adenovirus genome containing the ITR and thepackaging signal, an expression cassette with the gene of interest, anda portion of the adenovirus genome which can be used for homologousrecombination. In addition, adenovirus DNA is needed for recombinationwith the aforementioned adaptor plasmid. In the case of Ad-CMV-hcTK, theplasmid PCMV.TK was used as a basis. This plasmid contains nt. 1-455 ofthe adenovirus type 5 genome, nt. 456-1204 derived from pCMVβ (Clontech,the PstI-StuI fragment that contains the CMV enhancer promoter and the16S/19S intron from Simian Virus 40), the HSV TK gene (described in EPOPatent application 95202213), the SV40-derived polyadenylation signal(nt 2533-2668 of the SV40 sequence), followed by the BglII-ScaI fragmentof Ad5 (nt. 3328-6092 of the Ad5 sequence). These fragments are presentin a pMLP10-derived (Levrero et al., 1991) backbone. To generate plasmidpAD-CMVhc-TK, plasmid pCMV.TK was digested with ClaI (the uniqueClaI-site is located just upstream of the TK open readingframe) anddephosphorylated with Calf-Intestine Alkaline Phosphate. To generate ahairpin-structure, the synthetic oligonucleotides HP/clal (SEQ ID NO:19) and HP/cla2 (SEQ ID NO: 20) were annealed and phosphorylated ontheir 5-OH groups with T4-polynucleotide kinase and ATP. Thedouble-stranded oligonucleotide was ligated with the linearized vectorfragment and used to transform E. coli strain “Sure”. In section of theoligonucleotide into the ClaI site will disrupt the ClaI recognitionsites. The oligonucleotide contains a new ClaI site near one of itstermini. In selected clones, the orientation and the integrity of theinserted oligonucleotide was verified by sequence analyses. A clonecontaining the oligonucleotide in the correct orientation (the ClaI siteat the ITR side) was denoted pAd-CMV-hcTK. This plasmid wasco-transfected with ClaI digested wild-type Adenovirus-type5 DNA into911 cells. A recombinant adenovirus in which the CMV-hcTK expressioncassette replaces the E1 sequences was isolated and propagated usingstandard procedures.

To study whether the hairpin can be used as a primer for reverse strandsynthesis on the displaced strand after replication had started at theITR, the plasmid pICLhac is introduced into 911 cells (human embryonicretinoblasts transformed with the adenovirus E1 region). The plasmidpICLhaw serves as a control, which contains the oligonucleotide pairHP/asp1 (SEQ ID NO: 17) and 2 (SEQ ID NO: 18) in the reverse orientationbut is further completely identical to plasmid pICLhac. Also included inthese studies are plasmids pICLI and pICL. In the plasmid pICLI thehairpin is replaced by an adenovirus ITR. Plasmid pICL contains neithera hairpin nor an ITR sequence. These plasmids serve as controls todetermine the efficiency of replication by virtue of theterminal-hairpin structure. To provide the viral products other than theE1 proteins (these are produced by the 911 cells) required for DNAreplication the cultures are infected with the virus IG.Ad.MLPI.TK aftertransfection. Several parameters are being studied to demonstrate properreplication of the transfected DNA molecules. First, DNA extracted fromthe cell cultures transfected with aforementioned plasmids and infectedwith IG.Ad.MLPI.TK virus is being analyzed by Southern blotting for thepresence of the expected replication intermediates, as well as for thepresence of the duplicated genomes. Furthermore, virus is isolated fromthe transfected and IG.Ad.MLPI.TK infected cell populations, that iscapable of transferring and expressing a luciferase marker gene intoluciferase negative cells.

Plasmid DNA of plasmids pICLhac, pICLhaw, pICLI and pICL have beendigested with restriction endonuclease SalI and treated with mungbeannuclease to remove the 4 nucleotide single-stranded extension of theresulting DNA fragment. In this manner, a natural adenovirus 5′ ITRterminus on the DNA fragment is created. Subsequently, both the pICLhacand pICLhaw plasmids were digested with restriction endonuclease Asp718to generate the terminus capable of forming a hairpin structure. Thedigested plasmids are introduced into 911 cells, using the standardcalcium phosphate co-precipitation technique, four dishes for eachplasmid. During the transfection, for each plasmid two of the culturesare infected with the IG.Ad.MLPI.TK virus using 5 infectiousIG.Ad.MLPI.TK particles per cell. At twenty-hours post-transfection andforty hours post-transfection one Ad.tk-virus-infected and oneuninfected culture are used to isolate small molecular-weight DNA usingthe procedure devised by Hirt. Aliquots of isolated DNA are used forSouthern analysis. After digestion of the samples with restrictionendonuclease EcoRI using the luciferase gene as a probe a hybridizingfragment of approx. 2.6 kb is detected only in the samples from theadenovirus infected cells transfected with plasmid pICLhac. The size ofthis fragment is consistent with the anticipated duplication of theluciferase marker gene. This supports the conclusions that the insertedhairpin is capable to serve as a primer for reverse strand synthesis.The hybridizing fragment is absent if the IG.Ad.MLPI.TK virus isomitted, or if the hairpin oligonucleotide has been inserted in thereverse orientation.

The restriction endonuclease DpnI recognizes the tetranucleotidesequence 5′-GATC-3′, but cleaves only methylated DNA, (that is, onlyplasmid DNA propagated in, and derived, from E. coli, not DNA that hasbeen replicated in mammalian cells). The restriction endonuclease MboIrecognizes the same sequences, but cleaves only unmethylated DNA (viz.DNA propagated in mammalian cells). DNA samples isolated from thetransfected cells are incubated with MboI and DpnI and analyzed withSouthern blots. These results demonstrate that only in the cellstransfected with the PICLhac and the pICLI plasmids large DpnI-resistantfragments are present, that are absent in the MboI treated samples.These data demonstrate that only after transfection of plasmids pICLIand pICLhac replication and duplication of the fragments occur.

These data demonstrate that in adenovirus-infected cells linear DNAfragments that have on one terminus an adenovirus-derived ITR and at theother terminus a nucleotide sequence that can anneal to sequences on thesame strand, when present in single-stranded form thereby generate ahairpin structure, and will be converted to structures that haveinverted terminal repeat sequences on both ends. The resulting DNAmolecules will replicate by the same mechanism as the wild typeadenovirus genomes.

Experiment Series 2

The following demonstrates that the DNA molecules that contain aluciferase marker gene, a single copy of the ITR, the encapsidationsignal and a synthetic DNA sequence, that is capable of forming ahairpin structure, are sufficient to generate DNA molecules that can beencapsidated into virions.

To demonstrate that the above DNA molecules containing two copies of theCMV-luc marker gene can be encapsidated into virions, virus is harvestedfrom the remaining two cultures via three cycles of freeze-thaw crushingand is used to infect murine fibroblasts. Forty-eight hours afterinfection, the infected cells are assayed for luciferase activity. Toexclude the possibility that the luciferase activity has been induced bytransfer of free DNA, rather than via virus particles, virus stocks aretreated with DNaseI to remove DNA contaminants. Furthermore, as anadditional control, aliquots of the virus stocks are incubated for 60minutes at 56° C. The heat treatment will not affect the contaminatingDNA, but will inactivate the viruses. Significant luciferase activity isonly found in the cells after infection with the virus stocks derivedfrom IG.Ad.MLPI.TK-infected cells transfected with the pICLhc and pICLIplasmids. In neither the non-infected cells nor the infected cellstransfected with the pICLhw and pICL can significant luciferase activitybe demonstrated. Heat inactivation, but not DNaseI treatment, completelyeliminates luciferase expression, demonstrating that adenovirusparticles, and not free (contaminating) DNA fragments are responsiblefor transfer of the luciferase reporter gene.

These results demonstrate that these small viral genomes can beencapsidated into adenovirus particles and suggest that the ITR and theencapsidation signal are sufficient for encapsidation of linear DNAfragments into adenovirus particles. These adenovirus particles can beused for efficient gene transfer. When introduced into cells thatcontain and express at least part of the adenovirus genes (viz. E1, E2,E4, and L, and VA), recombinant DNA molecules that consist of at leastone ITR, at least part of the encapsidation signal as well as asynthetic DNA sequence, that is capable of forming a hairpin structure,have the intrinsic capacity to autonomously generate recombinant genomeswhich can be encapsidated into virions. Such genomes and vector systemcan be used for gene transfer.

Experiment Series 3

The following demonstrates that DNA molecules which contain nucleotides3510-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lack the E1 protein-coding regions, the right-hand ITR and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion of the same strand of the DNA molecule whenpresent in single-stranded form other than the ITR, and as a result iscapable of forming a hairpin structure, can replicate in 911 cells.

In order to develop a replicating DNA molecule that can provide theadenovirus products required to allow the above mentioned ICLhac vectorgenome and alike minimal adenovectors to be encapsidated into adenovirusparticles by helper cells, the Ad-CMV-hcTK adenoviral vector has beendeveloped. Between the CMV enhancer/promoter region and the thymidinekinase gene the annealed oligonucleotide pair HP/clal (SEQ ID NO: 19)and 2 (SEQ ID NO: 20) is inserted. The vector Ad-CMV-hcTK can bepropagated and produced in 911 cell using standard procedures. Thisvector is grown and propagated exclusively as a source of DNA used fortransfection. DNA of the adenovirus Ad-CMV-hcTK is isolated from virusparticles that had been purified using CsCl density-gradientcentrifugation by standard techniques. The virus DNA has been digestedwith restriction endonuclease ClaI. The digested DNA issize-fractionated on an 0.7% agarose gel and the large fragment isisolated and used for further experiments. Cultures of 911 cells aretransfected large ClaI-fragment of the Ad-CMV-hcTK DNA using thestandard calcium phosphate co-precipitation technique. Much like in theprevious experiments with plasmid plCLhac, the AD-CMV-hc will replicatestarting at the right-hand ITR. Once the 1-strand is displaced, ahairpin can be formed at the left-hand terminus of the fragment. Thisfacilitates the DNA polymerase to elongate the chain towards theright-hand-side. The process will proceed until the displaced strand iscompletely converted to its double-stranded form. Finally, theright-hand ITR will be recreated, and in this location the normaladenovirus replication-initiation and elongation will occur. Note thatthe polymerase will read through the hairpin, thereby duplicating themolecule. The input DNA molecule of 33250 bp, that had on one side anadenovirus ITR sequence and at the other side a DNA sequence that hadthe capacity to form a hairpin structure, has now been duplicated, in away that both ends contain an ITR sequence. The resulting DNA moleculewill consist of a palindromic structure of approximately 66500 bp.

This structure can be detected in low-molecular weight DNA extractedfrom the transfected cells using Southern analysis. The palindromicnature of the DNA fragment can be demonstrated by digestion of thelow-molecular weight DNA with suitable restriction endonucleases andSouthern blotting with the HSV-TK gene as the probe. This molecule canreplicate itself in the transfected cells by virtue of the adenovirusgene products that are present in the cells. In part, the adenovirusgenes are expressed from templates that are integrated in the genome ofthe target cells (viz. the E1 gene products), the other genes reside inthe replicating DNA fragment itself. Note however, that this linear DNAfragment cannot be encapsidated into virions. Not only does it lack allthe DNA sequences required for encapsidation, but also is its size muchtoo large to be encapsidated.

Experiment Series 4

The following demonstrates that DNA molecules which contain nucleotides3503-35953 (viz. 9.7-100 map units) of the adenovirus type 5 genome(thus lack the E1 protein-coding regions, the right-hand ITR and theencapsidation sequences) and a terminal DNA sequence that iscomplementary to a portion the same strand of the DNA molecule otherthan the ITR, and as a result is capable of forming a hairpin structure,can replicate in 911 cells and can provide the helper functions requiredto encapsidate the pICLI and pICLhac derived DNA fragments.

The following series of experiments aims to demonstrate that the DNAmolecule described in Experiment Series 3 could be used to encapsidatethe minimal adenovectors described in Experiment Series 1 and 2.

In the experiments the large fragment isolated after endonucleaseClaI-digestion of Ad-CMV-hcTK DNA is introduced into 911 cells (conformthe experiments described in part 1.3) together with endonuclease SalI,mungbean nuclease, endonuclease Asp718-treated plasmid pICLhac, or as acontrol similarly treated plasmid pICLhaw. After 48 hours virus isisolated by freeze-thaw crushing of the transfected cell population. Thevirus-preparation is treated with DNaseI to remove contaminating freeDNA. The virus is used subsequently to infect Rat2 fibroblasts.Forty-eight hours post infection, the cells are assayed for luciferaseactivity. Significant luciferase activity can be demonstrated only inthe cells infected with virus isolated from the cells transfected withthe pICLhac plasmid, and not with the pICLhaw plasmid. Heat inactivationof the virus prior to infection completely abolishes the luciferaseactivity, indicating that the luciferase gene is transferred by a viralparticle. Infection of 911 cell with the virus stock did not result inany cytopathological effects, demonstrating that the pICLhac is producedwithout any infectious helper virus that can be propagated on 911 cells.These results demonstrate that the proposed method can be used toproduce stocks of minimal-adenoviral vectors, that are completely devoidof infectious helper viruses that are able to replicate autonomously onadenovirus-transformed human cells or on non-adenovirus transformedhuman cells.

Besides the system described in this application, another approach forthe generation of minimal adenovirus vectors has been disclosed in PCTInternational Application WO 94/12649. The method described in WO94/12649 exploits the function of the protein IX for the packaging ofminimal adenovirus vectors (Pseudo Adenoviral Vectors (“PAV”) in theterminology of WO 94/12649). PAVs are produced by cloning an expressionplasmid with the gene of interest between the left-hand (including thesequences required for encapsidation) and the right-hand adenoviralITRs. The PAV is propagated in the presence of a helper virus.Encapsidation of the PAV is preferred compared the helper virus becausethe helper virus is partially defective for packaging. (Either by virtueof mutations in the packaging signal or by virtue of its size (virusgenomes greater than 37.5 kb package inefficiently). In addition, theauthors propose that in the absence of the protein IX gene the PAV willbe preferentially packaged. However, neither of these mechanisms appearto be sufficiently restrictive to allow packaging of only PAVs/minimalvectors. The mutations proposed in the packaging signal diminishpackaging, but do not provide an absolute block as the samepackaging-activity is required to propagate the helper virus. Alsoneither an increase in the size of the helper virus nor the mutation ofthe protein IX gene will ensure that PAV is packaged exclusively. Thus,the method described in WO 94/12649 is unlikely to be useful for theproduction of helper-free stocks of minimal adenovirus vectors/PAVs.

Although the application has been described with reference to certainpreferred embodiments and illustrative examples, the scope of theinvention is to be determined by reference to the appended claims.

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                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 22 <210> SEQ ID NO 1 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer Ea-1 <400> SEQUENCE: 1cgtgtagtgt atttataccc g            #                  #                   #21 <210> SEQ ID NO 2 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artifical #Sequence: Primer Ea-2 <400> SEQUENCE: 2tcgtcactgg gtggaaagcc a            #                  #                   #21 <210> SEQ ID NO 3 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer Ea-3 <400> SEQUENCE: 3tacccgccgt cctaaaatgg c            #                  #                   #21 <210> SEQ ID NO 4 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial  #Sequence:  Primer      Ea-5 <400> SEQUENCE: 4 tggacttgag ctgtaaacgc            #                   #                   # 20 <210> SEQ ID NO 5<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer Ep-2 <400> SEQUENCE: 5gcctccatgg aggtcagatg t            #                  #                   #21 <210> SEQ ID NO 6 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer Eb-1 <400> SEQUENCE: 6gcttgagccc gagacatgtc             #                  #                   # 20 <210> SEQ ID NO 7 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: <222> LOCATION:<223> OTHER INFORMATION: Description of Artificial #Sequence: Primer Eb-2 <400> SEQUENCE: 7cccctcgagc tcaatctgta tctt           #                  #                24 <210> SEQ ID NO 8 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence:  Primer       SV40-1 <400> SEQUENCE: 8gggggatccg aacttgttta ttgcagc           #                  #             27 <210> SEQ ID NO 9 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       SV40-2 <400> SEQUENCE: 9gggagatcta gacatgataa gatac           #                  #               25 <210> SEQ ID NO 10 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       Ad5-1 <400> SEQUENCE: 10gggagatctg tactgaaatg tgtgggc           #                  #             27 <210> SEQ ID NO 11 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer       Ad5-2 <400> SEQUENCE: 11ggaggctgca gtctccaacg gcgt           #                  #                24 <210> SEQ ID NO 12 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer ITR1 <400> SEQUENCE: 12gggggatcct caaatcgtca cttccgt           #                  #             27 <210> SEQ ID NO 13 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer ITR2 <400> SEQUENCE: 13ggggtctaga catcatcaat aatatac           #                  #             27 <210> SEQ ID NO 14 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: PCR primer       PCR/MLP1 <400> SEQUENCE: 14ggcgaattcg tcgacatcat caataatata cc        #                  #          32 <210> SEQ ID NO 15 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence:  PCT primer       PCR/MLP2 <400> SEQUENCE: 15ggcgaattcg gtaccatcat caataatata cc        #                  #          32 <210> SEQ ID NO 16 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: PCT primer       PCR/MLP3 <400> SEQUENCE: 16ctgtgtacac cggcgca              #                   #                  #   17 <210> SEQ ID NO 17 <211> LENGTH: 50 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: PCT primer       HP/asp1 <400> SEQUENCE: 17gtacactgac ctagtgccgc ccgggcaaag cccgggcggc actaggtcag  #              50 <210> SEQ ID NO 18 <211> LENGTH: 50 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence:  PCT primer       HP/asp2 <400> SEQUENCE: 18gtacctgacc tagtgccgcc cgggctttgc ccgggcggca ctaggtcagt  #              50 <210> SEQ ID NO 19 <211> LENGTH: 55 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: PCT primer       HP/cla1 <400> SEQUENCE: 19gtacattgac ctagtgccgc ccgggcaaag cccgggcggc actaggtcaa tc#gat          55 <210> SEQ ID NO 20 <211> LENGTH: 55 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:<222> LOCATION: <223> OTHER INFORMATION: Description of Artificial #Sequence: primer       HP/cla2 <400> SEQUENCE: 20gtacatcgat tgacctagtg ccgcccgggc tttgcccggg cggcactagg tc#aat          55 <210> SEQ ID NO 21 <211> LENGTH: 5620 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: Ad5 left terminus <222> LOCATION: 1..457<221> NAME/KEY: enhancer <222> LOCATION: 458..969 <221> NAME/KEY: exon<222> LOCATION: 970..1204 <221> NAME/KEY: gene<222> LOCATION: 1218..2987 <221> NAME/KEY: polyA_signal<222> LOCATION: 3018..3131 <221> NAME/KEY: pUC12 backbone<222> LOCATION: 3132..5620 <221> NAME/KEY: gene<222> LOCATION: 4337..5191<223> OTHER INFORMATION: Description of Artificial  #Sequence: Plasmid      pICL <400> SEQUENCE: 21catcatcaat aatatacctt attttggatt gaagccaata tgataatgag gg#ggtggagt     60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gc#ggaagtgt    120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt ga#cgtttttg    180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg ga#tgttgtag    240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aa#taagagga    300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gg#gccgcggg    360gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt tt#ccgcgttc    420cgggtcaaag ttggcgtttt attattatag tcaggggctg caggtcgtta ca#taacttac    480ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt ca#ataatgac    540gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tg#gagtattt    600acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cg#ccccctat    660tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga cc#ttatggga    720ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tg#atgcggtt    780ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc ca#agtctcca    840ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact tt#ccaaaatg    900tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gg#gaggtcta    960tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc ca#cgctgttt   1020tgacctccat agaagacacc gggaccgatc cagcctccgg actctagagg at#ccggtact   1080cgaggaactg aaaaaccaga aagttaactg gtaagtttag tctttttgtc tt#ttatttca   1140ggtcccggat ccggtggtgg tgcaaatcaa agaactgctc ctcagtggat gt#tgccttta   1200cttctagtat caagcttgaa ttcctttgtg ttacattctt gaatgtcgct cg#cagtgaca   1260ttagcattcc ggtactgttg gtaaaatgga agacgccaaa aacataaaga aa#ggcccggc   1320gccattctat cctctagagg atggaaccgc tggagagcaa ctgcataagg ct#atgaagaa   1380atacgccctg gttcctggaa caattgcttt tacagatgca catatcgagg tg#aacatcac   1440gtacgcggaa tacttcgaaa tgtccgttcg gttggcagaa gctatgaaac ga#tatgggct   1500gaatacaaat cacagaatcg tcgtatgcag tgaaaactct cttcaattct tt#atgccggt   1560gttgggcgcg ttatttatcg gagttgcagt tgcgcccgcg aacgacattt at#aatgaacg   1620tgaattgctc aacagtatga acatttcgca gcctaccgta gtgtttgttt cc#aaaaaggg   1680gttgcaaaaa attttgaacg tgcaaaaaaa attaccaata atccagaaaa tt#attatcat   1740ggattctaaa acggattacc agggatttca gtcgatgtac acgttcgtca ca#tctcatct   1800acctcccggt tttaatgaat acgattttgt accagagtcc tttgatcgtg ac#aaaacaat   1860tgcactgata atgaattcct ctggatctac tgggttacct aagggtgtgg cc#cttccgca   1920tagaactgcc tgcgtcagat tctcgcatgc cagagatcct atttttggca at#caaatcat   1980tccggatact gcgattttaa gtgttgttcc attccatcac ggttttggaa tg#tttactac   2040actcggatat ttgatatgtg gatttcgagt cgtcttaatg tatagatttg aa#gaagagct   2100gtttttacga tcccttcagg attacaaaat tcaaagtgcg ttgctagtac ca#accctatt   2160ttcattcttc gccaaaagca ctctgattga caaatacgat ttatctaatt ta#cacgaaat   2220tgcttctggg ggcgcacctc tttcgaaaga agtcggggaa gcggttgcaa aa#cgcttcca   2280tcttccaggg atacgacaag gatatgggct cactgagact acatcagcta tt#ctgattac   2340acccgagggg gatgataaac cgggcgcggt cggtaaagtt gttccatttt tt#gaagcgaa   2400ggttgtggat ctggataccg ggaaaacgct gggcgttaat cagagaggcg aa#ttatgtgt   2460cagaggacct atgattatgt ccggttatgt aaacaatccg gaagcgacca ac#gccttgat   2520tgacaaggat ggatggctac attctggaga catagcttac tgggacgaag ac#gaacactt   2580cttcatagtt gaccgcttga agtctttaat taaatacaaa ggatatcagg tg#gcccccgc   2640tgaattggaa tcgatattgt tacaacaccc caacatcttc gacgcgggcg tg#gcaggtct   2700tcccgacgat gacgccggtg aacttcccgc cgccgttgtt gttttggagc ac#ggaaagac   2760gatgacggaa aaagagatcg tggattacgt cgccagtcaa gtaacaaccg cg#aaaaagtt   2820gcgcggagga gttgtgtttg tggacgaagt accgaaaggt cttaccggaa aa#ctcgacgc   2880aagaaaaatc agagagatcc tcataaaggc caagaagggc ggaaagtcca aa#ttgtaaaa   2940tgtaactgta ttcagcgatg acgaaattct tagctattgt aatgggggat cc#ccaacttg   3000tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt ca#caaataaa   3060gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt at#cttatcat   3120gtctggatcg gatcgatccc cgggtaccga gctcgaattc gtaatcatgg tc#atagctgt   3180ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc gg#aagcataa   3240agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg tt#gcgctcac   3300tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc gg#ccaacgcg   3360cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact ga#ctcgctgc   3420gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta at#acggttat   3480ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag ca#aaaggcca   3540ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cc#tgacgagc   3600atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta ta#aagatacc   3660aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg cc#gcttaccg   3720gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tc#acgctgta   3780ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac ga#accccccg   3840ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac cc#ggtaagac   3900acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg ag#gtatgtag   3960gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga ag#gacagtat   4020ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt ag#ctcttgat   4080ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag ca#gattacgc   4140gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct ga#cgctcagt   4200ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg at#cttcacct   4260agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat ga#gtaaactt   4320ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tg#tctatttc   4380gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg ga#gggcttac   4440catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct cc#agatttat   4500cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca ac#tttatccg   4560cctccatcca gtctattaat tgtttgccgg aagctagagt aagtagttcg cc#agttaata   4620gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tc#gtttggta   4680tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cc#catgttgt   4740gcaaaaaagc ggttagctcc ttcggtgctc cgatcgttgt cagaagtaag tt#ggccgcag   4800tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg cc#atccgtaa   4860gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tg#tatgcggc   4920gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat ag#cagaactt   4980taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg at#cttaccgc   5040tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gc#atctttta   5100ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aa#aaagggaa   5160taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat ta#ttgaagca   5220tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aa#aaataaac   5280aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa ga#aaccatta   5340ttatcatgac attaacctat aaaaataggc gtatcacgag gcctatgcgg tg#tgaaatac   5400cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc ag#gctgcgca   5460actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gc#gaaagggg   5520gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cg#acgttgta   5580 aaacgacggc cagtgccaag cttgcatgcc tgcaggtcga     #                   #  5620 <210> SEQ ID NO 22 <211> LENGTH: 45<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<221> NAME/KEY: stem_loop <222> LOCATION: 11..45<223> OTHER INFORMATION: Description of Artificial  #Sequence: Asp 718      hairpin <400> SEQUENCE: 22gtacactgac ctagtgccgc ccgggcaaag cccgggcggc actag    #                  #45

What is claimed is:
 1. A recombinant nucleic acid molecule that can beacted on by a nucleic acid polymerase in a complementing cell comprisingat least the E1A gene of an adenovirus; wherein said recombinant nucleicacid molecule is a nucleic acid molecule based on or derived from anadenovirus, said recombinant nucleic acid molecule has at least onefunctional inverted terminal repeat, said recombinant nucleic acidmolecule comprises all other adenovirus derived genetic information notpresent in said complementing cell and necessary for replication, andsaid recombinant nucleic acid molecule is in a linear and essentiallysingle stranded form and comprises, at the molecule's 3′ terminus, arecombinantly fused sequence complementary to an upstream part of thesame strand of the recombinant nucleic acid molecule, to allow saidrecombinantly fused sequence and said upstream part to form base pairsand function as a start-site for said nucleic acid polymerase.
 2. Therecombinant nucleic acid molecule of claim 1, wherein said recombinantnucleic acid molecule has a functional inverted terminal repeat at eachterminus.
 3. The recombinant nucleic acid molecule of claim 1, whereinsaid recombinant nucleic acid molecule comprises a nucleic acid havingan adenovirus hr 400-404 mutation.
 4. The recombinant nucleic acidmolecule of claim 1, wherein said recombinant nucleic acid moleculecomprises an adenovirus E2A ts125 mutation.
 5. The recombinant nucleicacid molecule of claim 1, wherein said recombinant nucleic acid moleculecomprises an adenovirus E2 region under the control of an induciblepromoter.
 6. An isolated cell comprising the recombinant nucleic acidmolecule of claim
 1. 7. A methud of propagating a helper-dependentadenovirus in a complementing cell, comprising: providing therecombinant nucleic acid molecule of claim 1 to a complementing cell;and propagating the helper-dependent adenovirus in said complementingcell.
 8. The recombinant nucleic acid molecule of claim 1, wherein saidrecombinant nucleic acid molecule lacks overlapping sequences with thenucleic acid of the complementing cell into which it is transferred,said overlapping sequences otherwise enabling homologous recombinationleading to replication competent adenovirus in the complementing cell.9. The recombinant nucleic acid molecule of claim 1, wherein saidrecombinant nucleic acid molecule lacks a functional encapsidationsignal.